RATIONAL: AMP-activated protein kinase (AMPK) is an energy sensor and ubiquitously expressed in vascular cells. Recent studies suggest that AMPK activation improves endothelial function by counteracting oxidative stress in endothelial cells. How AMPK suppresses oxidative stress remains to be established. OBJECTIVE: The aim of this study is to examine the effects of AMPK in regulating NAD(P)H oxidase, oxidative stress, and endothelial function. METHODS AND RESULTS: The markers of oxidative stress, NAD(P)H oxidase subunit expression (gp91, p47, p67, NOX1 to-4), NAD(P)H oxidase-mediated superoxide production, 26S proteasome activity, IκBα degradation, and nuclear translocation of nuclear factor (NF)-κB (p50 and p65) were examined in cultured human umbilical vein endothelial cells and mouse aortas isolated from AMPKα2 deficient mice. Compared to the wild type, acetylcholine-induced endothelium-dependent relaxation was significantly impaired in parallel with increased production of oxidants in AMPKα2 ^-/- mice. Further, pretreatment of aorta with either superoxide dismutase (SOD) or tempol or apocynin significantly improved acetylcholine-induced endothelium-dependent relaxation in AMPKα2^-/- mice. Analysis of aortic endothelial cells from AMPKα2 ^-/- mice and human umbilical vein endothelial cells expressing dominant negative AMPK or AMPKα2-specific siRNA revealed that loss of AMPK activity increased NAD(P)H oxidase subunit expression (gp91, p47, p67, NOX1 and-4), NAD(P)H oxidase-mediated superoxide production, 26S proteasome activity, IκBα degradation, and nuclear translocation of NF-κB (p50 and p65), whereas AMPK activation by AICAR or overexpression of constitutively active AMPK had the opposite effect. Consistently, we found that genetic deletion of AMPKα2 in low-density lipoprotein receptor knockout (LDLr) strain markedly increased 26S proteasome activity, IκB degradation, NF-κB transactivation, NAD(P)H oxidase subunit overexpression, oxidative stress, and endothelial dysfunction, all of which were largely suppressed by chronic administration of MG132, a potent cell permeable proteasome inhibitor. CONCLUSIONS: We conclude that AMPKα2 functions as a physiological suppressor of NAD(P)H oxidase and ROS production in endothelial cells. In this way, AMPK maintains the nonatherogenic and noninflammatory phenotype of endothelial cells. Copyright © 2010 American Heart Association. All rights reserved.